Development of michrosystem technology suitable for bacterial identification and gene expression monitoring
As any phenotypical and taxonomical feature of living form is indexed in genome sequence and its expression, the aims of presented work were to develop appropriate DNA microarray formats suitable for both, sequence analysis at single-nucleotide accuracy and gene expression monitoring at the level of RNA transcription focussing on the actinomycete group of eubacteria. Both approaches were established in the course of work via optimization of the following parameters: solid support, its activation chemistry, structure and concentration of hybridization probes and targets, hybridization buffer, volume, time, washing conditions, signal detection and hybridization data analysis. Domain I of eubacterial 16S rRNA and its sequence signatures responsible for the taxonomical delineation within above group of bacteria at the genera level and above were chosen as taxonomical markers. PCR primers selected within conserved regions and oligonucleotide probes selected in the variability regions of taxonomical importance allowed a sensitive and specific detection of cognate bacterial sequences differing by a single base. A principal interchangeability of using either of the type of molecules, obtained PCR fragments or oligonucleotides, as attached probes and hybridisable targets, respectively, was shown. On the other hand, arrays composed of thousands spots carrying shotgun-library inserts of the erythromycin producer S. erythrea DSM40517 were constructed.. Hybridization with two RNA samples isolated from cells grown in rich and semisynthetic media was shown to result in technically plausible data with regard to specific and sensitive detection of genomic entities responding by perturbed expression rate to conditions investigated.